Bottom Line:
The degradation of Cdt1 by the CUL4-DDB1CDT2 E3 complex is an evolutionarily ancient pathway that is active in fungi and metazoa.In contrast, SCFSkp2-mediated Cdt1 degradation appears to have arisen relatively recently.A role for Skp2 in Cdt1 degradation has only been demonstrated in humans, and the pathway is not conserved in yeast, invertebrates, or even among other vertebrates.

ABSTRACTIn eukaryotes, DNA replication is strictly regulated so that it occurs only once per cell cycle. The mechanisms that prevent excessive DNA replication are focused on preventing replication origins from being reused within the same cell cycle. This regulation involves the temporal separation of the formation of the pre-replicative complex (pre-RC) from the initiation of DNA replication. The replication licensing factors Cdt1 and Cdc6 recruit the presumptive replicative helicase, the Mcm2-7 complex, to replication origins in late M or G1 phase to form pre-RCs. In fission yeast and metazoa, the Cdt1 licensing factor is degraded at the start of S phase by ubiquitin-mediated proteolysis to prevent the reassembly of pre-RCs. In humans, two E3 complexes, CUL4-DDB1CDT2 and SCFSkp2, are redundantly required for Cdt1 degradation. The two E3 complexes use distinct mechanisms to target Cdt1 ubiquitination. Current data suggests that CUL4-DDB1CDT2-mediated degradation of Cdt1 is S-phase specific, while SCFSkp2-mediated Cdt1 degradation occurs throughout the cell cycle. The degradation of Cdt1 by the CUL4-DDB1CDT2 E3 complex is an evolutionarily ancient pathway that is active in fungi and metazoa. In contrast, SCFSkp2-mediated Cdt1 degradation appears to have arisen relatively recently. A role for Skp2 in Cdt1 degradation has only been demonstrated in humans, and the pathway is not conserved in yeast, invertebrates, or even among other vertebrates.

Figure 2: The genesis of CUL4-DDB1CDT2 and SCFSkp2 E3 components. CUL4-DDBCDT2 and SCFSkp2 complex components were examined in representative organisms of diverse phyla (Table 2). A phylogenetic tree of the taxa analyzed, from eubacteria to mammals, is presented. Note that distances between branches are not to scale. Species and major classifications are color-coordinated, and the temporal locations of the presumed origins of E3 component genes are in red. CUL1-like and CUL4-like cullins, as well as their adaptor proteins DDB1 and Skp1, respectively, appear to have arisen early in eukaryotes, as they are absent from archaea and bacteria but are found in the eukaryotes examined. CDT2, the SRS for a CUL4-DDB1 E3 complex, appears to have arisen prior to the genesis of green plants. Skp2, the SRS for a CUL1 E3 complex, appears to have arisen after the genesis of fungi but prior to the genesis of metazoa. The branching order is based on a phylogenetic analysis using rRNA [76]. Note that other phylogenies, based on protein sequences, reverse the order of plants and slime molds [77]. Combining our genomic data with this alternative branching of phyla (not shown) would imply that CDT2 was created prior to plants in the main eukaryotic lineage but then lost within the slime mold lineage.

Mentions:
To determine when the genes for the CUL4-DDB1CDT2 and SCFSkp2 complexes arose during evolution, we analyzed divergent species using reciprocal BLAST searches [74]. We limited our analysis to those organisms in which the whole genome had been sequenced, so that a failure to detect a gene would be meaningful. Cullin genes were not found in bacteria or archaea, but at least two cullins were found in all of the eukaryotic genomes that we examined (Table 2). The observation of cullins in protists suggests that the cullin gene family arose early in the eukaryotic lineage (Table 2, Fig. 2). All eukaryotic species examined contain cullins that were most similar to metazoan CUL1 and CUL4 in reciprocal BLAST analysis, with the exception of budding yeast (which lacks a CUL4-like gene) (Table 2). This suggests that an ancestral duplication that gave rise to CUL1-like and CUL4-like genes occurred early in eukaryotic evolution. This result matches a phylogenetic analysis of cullins, in which the first branch point of the cullin phylogeny creates two clades, with the first clade giving rise to CUL1, CUL2 and CUL5, and the second clade giving rise to CUL3 and CUL4 [75]. The adaptor proteins Skp1 and DDB1 are present whenever CUL1-like and CUL4-like genes are observed, suggesting that the association between the cullins and their adaptor proteins is ancient (Table 2, Fig. 2).

Figure 2: The genesis of CUL4-DDB1CDT2 and SCFSkp2 E3 components. CUL4-DDBCDT2 and SCFSkp2 complex components were examined in representative organisms of diverse phyla (Table 2). A phylogenetic tree of the taxa analyzed, from eubacteria to mammals, is presented. Note that distances between branches are not to scale. Species and major classifications are color-coordinated, and the temporal locations of the presumed origins of E3 component genes are in red. CUL1-like and CUL4-like cullins, as well as their adaptor proteins DDB1 and Skp1, respectively, appear to have arisen early in eukaryotes, as they are absent from archaea and bacteria but are found in the eukaryotes examined. CDT2, the SRS for a CUL4-DDB1 E3 complex, appears to have arisen prior to the genesis of green plants. Skp2, the SRS for a CUL1 E3 complex, appears to have arisen after the genesis of fungi but prior to the genesis of metazoa. The branching order is based on a phylogenetic analysis using rRNA [76]. Note that other phylogenies, based on protein sequences, reverse the order of plants and slime molds [77]. Combining our genomic data with this alternative branching of phyla (not shown) would imply that CDT2 was created prior to plants in the main eukaryotic lineage but then lost within the slime mold lineage.

Mentions:
To determine when the genes for the CUL4-DDB1CDT2 and SCFSkp2 complexes arose during evolution, we analyzed divergent species using reciprocal BLAST searches [74]. We limited our analysis to those organisms in which the whole genome had been sequenced, so that a failure to detect a gene would be meaningful. Cullin genes were not found in bacteria or archaea, but at least two cullins were found in all of the eukaryotic genomes that we examined (Table 2). The observation of cullins in protists suggests that the cullin gene family arose early in the eukaryotic lineage (Table 2, Fig. 2). All eukaryotic species examined contain cullins that were most similar to metazoan CUL1 and CUL4 in reciprocal BLAST analysis, with the exception of budding yeast (which lacks a CUL4-like gene) (Table 2). This suggests that an ancestral duplication that gave rise to CUL1-like and CUL4-like genes occurred early in eukaryotic evolution. This result matches a phylogenetic analysis of cullins, in which the first branch point of the cullin phylogeny creates two clades, with the first clade giving rise to CUL1, CUL2 and CUL5, and the second clade giving rise to CUL3 and CUL4 [75]. The adaptor proteins Skp1 and DDB1 are present whenever CUL1-like and CUL4-like genes are observed, suggesting that the association between the cullins and their adaptor proteins is ancient (Table 2, Fig. 2).

Bottom Line:
The degradation of Cdt1 by the CUL4-DDB1CDT2 E3 complex is an evolutionarily ancient pathway that is active in fungi and metazoa.In contrast, SCFSkp2-mediated Cdt1 degradation appears to have arisen relatively recently.A role for Skp2 in Cdt1 degradation has only been demonstrated in humans, and the pathway is not conserved in yeast, invertebrates, or even among other vertebrates.

ABSTRACTIn eukaryotes, DNA replication is strictly regulated so that it occurs only once per cell cycle. The mechanisms that prevent excessive DNA replication are focused on preventing replication origins from being reused within the same cell cycle. This regulation involves the temporal separation of the formation of the pre-replicative complex (pre-RC) from the initiation of DNA replication. The replication licensing factors Cdt1 and Cdc6 recruit the presumptive replicative helicase, the Mcm2-7 complex, to replication origins in late M or G1 phase to form pre-RCs. In fission yeast and metazoa, the Cdt1 licensing factor is degraded at the start of S phase by ubiquitin-mediated proteolysis to prevent the reassembly of pre-RCs. In humans, two E3 complexes, CUL4-DDB1CDT2 and SCFSkp2, are redundantly required for Cdt1 degradation. The two E3 complexes use distinct mechanisms to target Cdt1 ubiquitination. Current data suggests that CUL4-DDB1CDT2-mediated degradation of Cdt1 is S-phase specific, while SCFSkp2-mediated Cdt1 degradation occurs throughout the cell cycle. The degradation of Cdt1 by the CUL4-DDB1CDT2 E3 complex is an evolutionarily ancient pathway that is active in fungi and metazoa. In contrast, SCFSkp2-mediated Cdt1 degradation appears to have arisen relatively recently. A role for Skp2 in Cdt1 degradation has only been demonstrated in humans, and the pathway is not conserved in yeast, invertebrates, or even among other vertebrates.